Nucleic acid testing often requires amplification of nucleic acids to achieve a sufficient concentration and/or purity to undergo subsequent testing. Sometimes amplification of nucleic acids is used as a surrogate in detection of non-nucleic acids, such as proteins. The majority of nucleic acid amplification/extension reactions depend on the presence of a primer comprised of modified or natural nucleic acids at the 3′ end which allow extension in the presence of a polymerase.
A universal problem with such amplification reactions is the presence of primer-dimers. Primer-dimers are formed when primers extend each other rather than the target nucleic acid. Primer-dimers use up primers, resulting in the presence of impurities in the reaction. Even worse, primer-dimers can use up enough primers to cause false negatives in some cases. Or, if interacting with a probe, primer-dimers can cause false positives.
A variety of hot starts have been developed to deal with the issue of primer-dimers including suspending the polymerase in a wax material, inhibiting the polymerase with antibodies, chemically modifying the polymerase, sequestering primers, and a variety of other methods. The problem with all of these methods is that they are only effective prior to the first round of amplification/extension. Any primer-dimers that form thereafter are amplified at an exponential rate.
Other methods of dealing with primer-dimers include methods such as nested PCR. However, this requires two separate reactions and increases the chances of contamination.
Many amplification/extension reactions are also coupled with a detection probe. The principles often revolve around a labeled linear probe, such as Taqman or a labeled hairpin probe such as Molecular Beacons. Some methods achieve incredible specificity through the use of cooperatively linking two probes, such as Tentacle Probes. However, each of these probe based methods is limited in detecting mutants in a high background of wild type. While they can achieve all or nothing detection of single nucleotide polymorphisms and other mutations, they can only pick out about one mutant in a background of 10 to 20 wild type sequences. This is because the primers amplify both the wild type and the mutant and are depleted without being able to detect both. Methods like ARMS can be combined with the probe detection technologies to overcome this problem to an extent, but cannot be effectively multiplexed for real-time detection when more than one mutation occurs in the same general region.
Several primers have been developed which include a detection mechanism, such as Amplifluor primers, Rapid Detex primers and Scorpion primers. The first two are especially prone to false positives from primer-dimer problems because they are not sequence specific. The latter is a self-probing primer, where the probe binds to the primer extension product rather than the nucleic acid template. Because it has a sequence specific probe, it is less likely to result in false positives, but is still subject to primer-dimer associated problems.